Role of PDK1 in pancreatic cancer cell fitness under tumor nutrient stress

PDK1在肿瘤营养应激下胰腺癌细胞健康中的作用

• 批准号: 10751747
• 负责人: Colin Sheehan
• 金额: $ 4.77万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-30 至 2025-09-29
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10751747
• 关键词: Affect; Anabolism; Animals; Biochemical; Biological Assay; Biological Markers; Biological Models; Blood Vessels; CRISPR interference; CRISPR screen; Carbon; Cell Survival; Cells; Citric Acid Cycle; Clinic; Communication; Data; Dependence; Disease; Drug Compounding; Environment; Equilibrium; Fermentation; Formulation; Genetic; Goals; Growth; Homeostasis; Hypoxia; Injections; Intercellular Fluid; Lactic acid; Lipids; Malignant neoplasm of pancreas; Mediating; Mediator; Metabolic; Metabolism; Modeling; Mus; NADH; Nucleic Acids; Nutrient; Nutrient availability; Oxidation-Reduction; Oxidative Stress; Oxygen; PDH kinase; Pancreatic Ductal Adenocarcinoma; Pathway interactions; Perfusion; Phosphorylation; Phosphotransferases; Production; Proliferating; Proteins; Proto-Oncogene Proteins c-akt; Pyruvate; Pyruvate Dehydrogenase Complex; Reactive Oxygen Species; Regulation; Role; Signal Pathway; Signal Transduction; Stress; Testing; Therapeutically Targetable; Tissues; Work; aerobic glycolysis; cancer cell; cancer type; cell growth; clinical translation; clinically relevant; cofactor; cytotoxic; effective therapy; fitness; in vivo; knock-down; macromolecule; metabolic abnormality assessment; metabolomics; neoplastic cell; normoxia; novel; novel therapeutic intervention; oxidation; pancreatic cancer cells; pancreatic ductal adenocarcinoma cell; pancreatic ductal adenocarcinoma model; prevent; pyruvate dehydrogenase; response; screening; sensor; therapeutic target; tumor; tumor growth; tumor microenvironment

PROJECT SUMMARY Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer type in urgent need of more effective treatment options. A notable feature of PDAC tumors is the highly altered nutrient conditions present within the tumor microenvironment (TME) caused by poor perfusion from the supporting tumor vasculature. Although PDAC cells can rapidly proliferate within these suboptimal nutrient conditions, the metabolic adaptations they rely upon to do so remain unknown. Furthermore, understanding these adaptations has the potential to reveal therapeutically targetable vulnerabilities of PDAC cells in vivo. Towards this end, we have developed a novel medium formulation (TIFM) that recapitulates the nutrient conditions present within PDAC tumors in vivo, in order to study the metabolic responses of PDAC cells to tumor nutrient stress using tractable ex vivo models. Applying pooled CRISPR-interference (CRISPRi) screening to this new model system, we surprisingly identified a hypoxia-induced kinase, pyruvate-dehydrogenase kinase 1 (PDK1), as being critical to the fitness of PDAC cells in TIFM, even under normoxia conditions. PDK1 is a kinase normally activated by hypoxia to inhibit the pyruvate dehydrogenase (PDH) complex and thus redirect pyruvate-derived carbon away from the TCA cycle and towards lactic acid fermentation. The overall goal of this proposal is to understand how and why PDK1 and aerobic glycolysis become critical to PDAC cellular fitness during tumor nutrient stress, and to evaluate in vivo the functional dependence of PDAC cells on suppressed pyruvate oxidation for tumor growth. I hypothesize that two potential mechanisms may underlie PDK1 dependency in TIFM: (1) PDK1 promotes aerobic glycolysis to prevent the production of cytotoxic levels of reactive oxygen species (ROS) or (2) supports NAD+/NADH cofactor balance for macromolecule biosynthesis. Further, I hypothesize that PDK1 activity is upregulated under TIFM conditions through a nutrient-sensitive mTORC2-AKT-PDK1 signaling axis. We will investigate this hypothesis in three specific aims. Aim 1. Functional and metabolic assays will be performed to determine the adaptive function of PDK1 under tumor nutrient stress. Aim 2. Functional and biochemical assays will be performed to identify the nutrient factor in TIFM responsible for PDK1 dependency and elucidate the signaling pathway that communicates its availability to the TCA cycle. Aim 3. Animal studies will be performed to determine the essentiality of suppressed pyruvate oxidation for PDAC tumor growth in vivo. These findings reveal a critical metabolic dependency of cancer cells in adaptation to the nutrient stress present in the TME. Furthermore, understanding this adaptation may reveal novel therapeutic strategies for managing pancreatic cancer in the clinic based on metabolic constraints set by the tumor microenvironment.

项目概要 胰腺导管腺癌（PDAC）是一种高度侵袭性的癌症类型，迫切需要更多的治疗 有效的治疗方案。 PDAC 肿瘤的一个显着特征是存在高度改变的营养条件 肿瘤微环境（TME）内由支持肿瘤脉管系统的灌注不良引起。 尽管 PDAC 细胞可以在这些次优营养条件下快速增殖，但代谢 他们所依赖的适应能力仍然未知。此外，了解这些适应也有助于 揭示 PDAC 细胞体内治疗靶向脆弱性的潜力。为此，我们有 开发了一种新型培养基配方 (TIFM)，它概括了 PDAC 中存在的营养条件 体内肿瘤，以便使用易处理的方法研究 PDAC 细胞对肿瘤营养应激的代谢反应 离体模型。将混合 CRISPR 干扰 (CRISPRi) 筛选应用于这个新模型系统，我们 令人惊讶的是，我们发现了一种缺氧诱导的激酶，丙酮酸脱氢酶激酶 1 (PDK1)，对于 即使在常氧条件下，PDAC 细胞在 TIFM 中的适应性。 PDK1 是一种激酶，通常由 缺氧会抑制丙酮酸脱氢酶（PDH）复合物，从而将丙酮酸衍生的碳重定向 从 TCA 循环到乳酸发酵。该提案的总体目标是了解如何 以及为什么 PDK1 和有氧糖酵解在肿瘤营养应激期间对 PDAC 细胞健康至关重要，以及 体内评估 PDAC 细胞对抑制肿瘤丙酮酸氧化的功能依赖性 生长。我假设 TIFM 中 PDK1 依赖性可能有两种潜在机制：(1) PDK1 促进有氧糖酵解，以防止产生细胞毒性水平的活性氧 (ROS) 或 (2) 支持大分子生物合成的 NAD+/NADH 辅因子平衡。此外，我假设 PDK1 在 TIFM 条件下，活性通过营养敏感的 mTORC2-AKT-PDK1 信号轴上调。 我们将从三个具体目标来研究这一假设。目标 1. 功能和代谢测定将 进行以确定PDK1在肿瘤营养应激下的适应性功能。目标 2. 功能性和 将进行生化检测以确定 TIFM 中导致 PDK1 依赖的营养因子 并阐明将其可用性传达给 TCA 循环的信号通路。目标 3. 动物研究 将进行以确定抑制丙酮酸氧化对 PDAC 肿瘤生长的重要性 体内。这些发现揭示了癌细胞在适应营养应激方面的关键代谢依赖性 存在于 TME 中。此外，了解这种适应可能会揭示新的治疗策略 根据肿瘤微环境设定的代谢限制在临床上管理胰腺癌。